Turin, June 21-24, 2011

IN VITRO VITAMIN K TREATMENT IS NOT CAPABLE TO RESCUE UNEFFICIENT MGP-CARBOXYLATION IN PXE FIBROBLASTSAnnovi G., Boraldi F., Quaglino D.Dept. Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy

Phylloquinone (vitamin K1)

Menaquinone (vitamin K2)

The inappropriate biomineralization occurring in soft tissue is defined as

ectopic calcification

• atherosclerosis• chronic renal disease• diabetes• treatment with anti-coagulant therapy• specific gene defects

pseudoxanthoma elasticum

Ectopic calcification occurence:

1. Circulating nucleational complexes (high Ca / P)2. Cell death3. Alteration of NF-kB activity4. Proteolysis and ECM degradation5. Presence of “Bone Proteins”6. Loss of inhibitors

a 14-kDa secreted protein containing 5-glutamic acid residues that must be γ-carboxylated by a vitamin K-dependent γ-carboxylase in order to acquire calcium-binding properties.

Matrix Gla Protein (MGP)

-COOH

Vit. K1H2

CO2 + O2

Vit. K1

VK

OR

Vit. K1>O

Vitamin K CycleGamma-

carboxylase

MGP

MGP

GLA

GLA

GLA GLA GLA

Lab Invest. 2010 Jun;90(6):895-905. Epub 2010 Apr 5.

Low serum vitamin K in PXE results in defective carboxylation of mineralization inhibitors similar to the GGCX mutations in the PXE-like syndrome.

Vanakker OM, Martin L, Schurgers LJ, Quaglino D, Costrop L, Vermeer C, Pasquali-Ronchetti I, Coucke PJ, De Paepe A.

Serum Gla MGP

%

Serum Vit Kng/ml

40 proteins differentiallyexpressed between control andPXE fibroblasts

CO2 + O2

Vit. K1H2

Vit. K1>O

Vitamin K cycle

Vit. K1

VK

OR

gamma-carboxylase

-COOHMGP

MGP

CALUCALU

e-

PDI

SH SH

PDI

S S

SHSHs s

50

37

0

40

80

120

160 *

CALU

PDI

75

50

C PXE

Control PXE K1 100µM K1 100µM K1 1µM K1 1µM K1 0.01µMK1 0.01µM0.00

0.25

0.50

0.75

1.00

1.25

1.50 Controllo PXE

*

*p<0.05 PXE vs ControlA

rbit

rary

un

it D

H2

Control PXE K2 100µM K2 100µM K2 1µM K2 1µM K2 0.01µMK2 0.01µM0.00

0.25

0.50

0.75

1.00

1.25

Control PXE

K2 100 µM K2 1 µM

* p<0.05 PXE vs Control$ p<0.05 Vitamin K2 PXE vs PXE

$ $

*

Arb

itra

ry u

nit

DH 2

Control PXE K1 100µM K1 100µM K1 1µM K1 1µM K1 0.01µMK1 0.01µM0.0

0.5

1.0

1.5

2.0

Control PXE

Arb

itra

ry u

nit

H2O

2

Control PXE K2 100µM K2 100µM K2 1µM K2 1µM K2 0.01µMK2 0.01µM0.0

0.5

1.0

1.5

2.0

K2 100 µM K2 1 µM K2 0.01 µM

Control PXE

Arb

itra

ry u

nit

H2O

2

0.1µM 100µM

Vitamin K2

UT DMF UT DMF 0.1µM 100µM 0.1µM 100µM

Vitamin K1

0.1µM 100µM

1± 0

.15

0.97

± 0.

29

1.2

± 0.2

5

0.94

± 0.

1

1.06

± 0.

131.

34 ±

0.32

1.70

± 0.

51

0.99

± 0.

15

1.62

± 0.

33

1.10

± 0

.11

1.07

± 0.

491.

42 ±

0.27

Control Control ControlPXE PXEPXE

Control PXE Control PXE0.0

0.5

1.0

1.5

*

*

Rel

ativ

e fo

ld c

han

ges

PDI CALU

DMFDMFK1 0.01mMK1 0.01mMK1 100mMK1 100mM0,1 0,1 100 1000.0

0.5

1.0

1.5 Control PXE

Rel

ativ

e fo

ld c

han

ges

(CA

LU

)

100 µM 0.1 µMDMF 100 µM 0.1 µM

**

** *

** *

Vit K1 Vit K2

DMFDMFK1 0.01mMK1 0.01mMK1 100mMK1 100mMK2 0.01mMK2 0.01mMK2 100mMK2 100mM0.0

0.5

1.0

1.5

2.0

100µM 0.1µMDMF

ControlPXE

100µM 0.1µM

Rela

tive f

old

ch

an

ges (

PD

I)

Vit K1 Vit K2

DMF CDMF P0.1 K10.1K1100 K1100 K10.1 K20.1 K2100 K2 C100 K2 P0

1

2

3

100 µM 0.1 µMDMF 100 µM 0.1 µM

* *

ControlPXE

Rel

ativ

e m

RN

A f

old

ch

ang

e

Solv Solv0,1µM0,1µM100µM100µM0,1M0,1mM100M100M0.0

0.5

1.0

1.5 Control PXE

Rel

ativ

e fo

ld c

han

ges

(MG

P)

100 µM 0.1 µMDMF 100 µM 0.1 µM

*

*

*

Vit K1 Vit K2

0

1

2

3

100µM0,1µM 100µM0,1µM

Control PXE

Gla

-MG

P (

a.u

.)

Vitamin K1 Vitamin K2

Control PXE

100µ

M0.

1µM

Vitamin K2

100µ

M0.

1µM

Vitamin K1

Control PXE

0,1µM K2100µM K10,1µM K1 100µM K2DMF

Con

trol

PX

E

Days of culture DMF Vitamin K1 Vitamin K2

0.1µM 100µM 0.1µM 100µM

Day 10

Control 0.2 ±0.20 0.4 ±0.25 0.6 ±0.40 0.7 ±0.66 0.8 ±0.48

PXE 0.2 ±0.17 0.4 ±0.40 0.8 ±0.58 0.3 ±0.28 0.4 ±0.24

Day 20

Control 0.3 ±0.25 1.4 ±0.87 2.6 ±1.39 0.7 ±0.67 2.0 ±0.81 #

PXE 1.2 ±0.56 1.8 ±1.11 4.6 ±2.64 3.4 ±2.57 1.7 ±1.66 #

Day 30

Control 9.9±3.28 9.4 ±5.01 9.0 ±5.81 26.0 ±7.72 n.d.b)

PXE 14.4 ±4.15 12.0±4.3 11.9±3.86 22.2 ±5.76 n.d. b)

# morphological alteration

In these work we have demontrated that:

# PXE fibroblasts are able to respond to vitamin K increasing the efficiency of the carboxylation process.

# vitamin K2 reduce the expression of MGP mRNA and protein.

#vitamin k have negligible effects on MGP carboxylation

#vitamin k are not able to counteract the in vitro mineralization process.

° mice data°in vivo data